Peroxidase is an enzyme that catalyzes the oxidation of various compounds, such as phenols and amines, by peroxides. In addition, particular compounds have been termed pseudoperoxidases because these compounds behave in a manner similar to the peroxidase enzyme. Accordingly, pseudoperoxides liberate oxygen from hydroperoxides and transfer the oxygen to certain acceptor compounds. Therefore, in general, the pseudoperoxidases are enzyme-like in that they catalyze, or otherwise participate in, reactions between peroxides and oxidizable compounds. The pseudoperoxidases also are termed peroxidatively active substances, and include hemoglobin and its derivatives.
For example, in the assay of urine for glucose, glucose oxidase, in the presence of oxygen, first converts the glucose in the urine to gluconic acid and hydrogen peroxide. Then, the peroxidase enzyme, also present in the assay, catalyzes the interaction between the hydrogen peroxide and an oxidizable dye compound, like o-tolidine. The dye compound, usually essentially colorless in its reduced state, undergoes a color transition upon oxidation, such as to a blue color for o-tolidine, by the peroxidase-catalyzed interaction with hydrogen peroxide. The degree and intensity of the color transition are directly proportional to the amount of hydrogen peroxide generated by the glucose conversion. Then, the amount of hydrogen peroxide generated by the glucose conversion is correlated to the original concentration of glucose in the urine sample.
Similarly, a peroxidatively active substance, like hemoglobin and its derivatives, catalyzes the interaction between a hydroperoxide and an oxidizable dye. In such interactions, the peroxidatively active substance imitates the peroxidase enzyme, and catalyzes or otherwise participates in an interaction between the oxidizable dye and the hydroperoxide. The oxygen liberated from a hydroperoxide by a peroxidatively active substance is transferred to an oxidizable dye. The resulting interaction provides a detectable response, such as a color transition, wherein the intensity of the response is indicative of the presence or the concentration of the peroxidatively active substance.
Assays for a peroxidatively active substance are based upon the above-described chromogenic interaction, wherein the degree and intensity of the color transition of the indicator dye are correlated to the concentration of the peroxidatively active substance in the test sample. Assays for a peroxidatively active substance are particularly useful in detecting and measuring low concentrations of blood, often termed "occult" blood, in body fluid samples such as urine, feces or gastrointestinal contents. Although occult blood in urine, feces or vomit usually is not visible to the naked eye, the detection of occult blood is important in the diagnosis of hemorrhages in the stomach, intestines and urinary tract. The hemorrhages are caused, for example, by tumors, ulcers or inflammations of the organ in question. Presently, most methods of determining the presence of occult blood in a test sample are based upon the pseudoperoxidase activity of hemoglobin or myoglobin.
Although protein in urine is the most important indicator of renal dysfunction, the presence of blood in urine also is an indication of damage to the kidney or urinary tract. Normally, detectable amounts of occult blood are not present in urine, even with very sensitive chemical methods. Blood in the urine can appear as intact red blood cells or as free hemoglobin. Usually the presence of free hemoglobin indicates that the blood cells have ruptured either because of a traumatic passage through the kidney and urinary tract to the bladder, or because the blood cells have been exposed to dilute urine in the bladder that caused the cells to hemolyze.
More particularly, the presence of blood in urine or feces is a symptom of a variety of abnormal conditions, including cancer. The presence of blood in urine, as indicated by a positive test for occult blood, often indicates bleeding in the urinary tract. Free hemoglobin is present in the urine because of renal disorders, infectious diseases, neoplasms, or traumas affecting part of the urinary tract. Free hemoglobin in the urine also can indicate a transfusion reaction, hemolytic anemia, or paroxysmal hemoglobinuria, or can appear from various poisonings or following severe burns. In addition, a positive chemical test for hemoglobin, without the presence of red cells, can indicate myoglobinuria as a result of traumatic muscle injury.
Hemoglobinuria is defined as the presence of free hemoglobin in the urine without red blood cells. In contrast, hematuria is defined as the presence of intact red blood cells in urine. Hematuria is indicative of a specific defect in the microscopic functional unit (the nephron) of the kidney, and is indicative of bleeding in the kidney, the ureter, the bladder or the urethra. The free hemoglobin in the plasma is excreted by the kidney into the urine. In some situations, hemolysis of the red blood cells occurs after the cells have entered the urine. Most urine samples containing red blood cells also contain some hemolyzed occult blood. Presently, the differentiation of trace amounts of blood as cells versus free hemoglobin is of little significance.
Myoglobin, the red respiratory pigment of muscle tissue, is another peroxidatively active substance. Myoglobin is very similar to hemoglobin in its composition and chemical reactions. Myoglobin can be liberated from muscle cells by certain types of injury, and in such cases the myoglobin will circulate in the plasma, and then be excreted in the urine. In addition, certain genetic muscle disorders can cause the muscles to lose myoglobin that subsequently appears in the urine. Myoglobin also is found in the urine after a cardiac infarct.
Hematuria, hemoglobinuria or myoglobinuria depends upon the precise nature of the clinical and pathological disorder and upon the severity of the specific disease or injury. In addition, other peroxidatively active substances also are present in leukocytes and bacteria. Overall, the detection of a peroxidatively active substance is especially important in the diagnosis of diseases and infections of the kidneys and urinary tract.
Therefore, accurate and thorough assays of urine and other test samples for peroxidatively active substances must be available for both laboratory and home use. The assays must permit the detection and measurement of the peroxidatively active substance such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the assay method could be utilized in a dip-and-read format for the easy and economical, qualitative or quantitative determination of a peroxidatively active substance in urine or other rest samples.
Furthermore, any method of assaying for a peroxidatively active substance in urine or other test sample must yield accurate, trustworthy and reproducible results by utilizing an indicator reagent composition that undergoes a color transition as a result of an interaction with a peroxidatively active substance, and not as a result of a competing chemical or physical interaction, such as a preferential interaction with a test sample component other than a peroxidatively active substance or a color transition occurring due to the instability of the indicator reagent composition. Moreover, it would be advantageous if the assay method for a peroxidatively active substance is suitable for use in dry phase reagent strips for the rapid, economical and accurate determination of a peroxidatively active substance in urine or other test sample. Additionally, the method and composition utilized in the assay for a peroxidatively active substance should not adversely affect or interfere with the other test reagent pads that are present on multideterminant reagent strips.
Therefore, in order to determine if an individual is excreting a peroxidatively active substance, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive detection assays for a peroxidatively active substance, like occult blood, have been developed. Furthermore, of the several different assay methods developed for the detection or measurement of occult blood in urine, the methods based on dip-and-read dry phase test strips have proven especially useful because dry phase test strip methods are readily automated and provide reproducible and accurate results.
Some tests strips used in assays for peroxidatively active substances have a single test area consisting of a small square pad of a suitable carrier matrix impregnated with an indicator reagent composition comprising an indicator dye, such as a benzidine dye; a hydroperoxide; and a buffer. Other test strips are multideterminant reagent strips that include one test area for the assay of a peroxidatively active substance as described above, and further include several additional test areas on the same strip to permit the simultaneous assay of other urinary constituents. For both types of colorimetric test strips, the assay for a peroxidatively active substance in urine is performed simply by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the colorimetric test strip bottle. Occult blood tests usually are included on multideterminant reagent strips to screen urine samples during routine physical examinations because it is important to detect a bleeding condition early.
The test strip method is the simplest and most direct assay for the presence of blood in urine. The test area is impregnated with an oxidizable indicator dye, like 3,3',5,5'-tetramethylbenzidine, and a buffered hydroperoxide. The test area becomes a green to dark blue color when hemoglobin present in the urine sample catalyzes the oxidation reaction of tetramethylbenzidine by the hydroperoxide. The development of green spots on the tests area indicates intact, nonhemolyzed erythrocytes. In accordance with the above-described method, an individual can readily determine, visually, the concentration of a peroxidatively active substance in a urine sample. The color of the test strip is compared with a color chart approximately one minute after the test strip is dipped into the urine. The color blocks on the color chart indicate negative, nonhemolyzed trace, hemolyzed trace, small (1+), moderate (2+), and large (3+) amounts of blood. The color chart ranges from orange through green to blue. The assay usually is capable of detecting from about 0.015 to about 0.06 mg/dL (milligrams per deciliter) of free hemoglobin or from about 5 to about 20 intact red blood cells per microliter.
In addition, ascorbate ion, when present, seriously interferes in the above-described assay method for a peroxidatively active compound. It has been found that including certain metal ion complexes in the indicator reagent composition essentially eliminate the ascorbate interference problem. However, in general, the metal ion complexes also demonstrate peroxidase activity, and behave similarly to peroxidase or the pseudoperoxidases to catalyze the color-forming reaction between a hydroperoxide and a oxidizable dye. Accordingly, although the metal ion complexes eliminate ascorbate interference, the metal ion complexes also can produce false positive assays because the metal ion complexes can catalyze oxidation of the oxidizable dye by the hydroperoxide, thereby producing a color change in the device even though a peroxidatively active substance is not present in the test sample.
Investigators have found that particular ferric ion complexes substantially reduced the false positive assay results attributed to most metal ion complexes used to eliminate ascorbate interference. However, although the ferric ion complexes effectively eliminated ascorbate interferences and demonstrated a substantially reduced peroxidative activity, the indicator reagent composition had to be buffered to a pH value that does not provide the optimum color transition.
Therefore, it would be extremely advantageous to provide a simple, accurate and trustworthy method of assaying urine for low levels of a peroxidatively active substance. Present day test strips for a peroxidatively active substance incorporate an indicator reagent composition that includes an amine borate stabilizer. However, the amine borates used in the prior art have the disadvantage of hydrolyzing upon exposure to environmental humidity, resulting in an increased pH of the indicator reagent composition. Consequently, the present day indicator reagent compositions, also including a metal ion complex for ascorbate resistance and buffered at a pH that does not provide the most spectacular color transition, increase in pH due to hydrolysis of the amine borates. Therefore, the sensitivity of the assay for a peroxidatively active substance is decreased. Surprisingly and unexpectedly, the method of the present invention essentially eliminates an increase in pH of the indicator reagent composition after exposure to humid conditions by including a hydrolysis-resistant amine borate in the indicator reagent composition. Therefore, the problem of decreased assay sensitivity is solved, even in the presence of a compound, like a metal ion complex, to eliminate ascorbate interference.
Accordingly, a quantitative urine assay for a peroxidatively active substance can be performed by laboratory personnel to afford immediate and trustworthy test results by providing a more accurate assay method in an easy-to-use form, such as a dip-and-read test strip. In addition, the test strip method can be performed by the patient at home to more precisely monitor the level of a peroxidatively active substance in urine and/or the success of the medical treatment the patient is undergoing.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy assay for a peroxidatively active substance by utilizing a test strip that includes a test pad comprising a suitable carrier matrix impregnated with an indicator reagent composition of the present invention. The indicator reagent composition comprises an indictor dye; a hydroperoxide; a bicyclic amine borate compound that resists hydrolysis upon exposure to humid conditions; and a buffer. The indicator reagent composition is sensitive to low concentrations of a peroxidatively active substance; stabilizes the indicator dye and thereby essentially eliminates a premature interaction between the indicator dye and a hydroperoxide; and, surprisingly and unexpectedly, essentially eliminates an increase in the pH of the test strip upon exposure to humid conditions. Accordingly, the improved stability of the indicator reagent composition enhances the sensitivity of the assay, thereby providing a more accurate and trustworthy assay for a peroxidatively active substance.
Prior to the present invention, no known method of assaying urine or other test samples for peroxidatively active substances included an indicator reagent composition comprising an indicator dye; a hydroperoxide; a buffer; and a hydrolysis-resistant amine borate that stabilizes the indicator dye of the indicator reagent composition. Although a dry phase test strip including an oxidizable indicator dye, such as o-tolidine or 3,3.dbd.,5,5'-tetramethylbenzidine; a buffer; and a hydroperoxide has been used previously, dry phase test strips incorporating these three compounds demonstrated a tendency to undergo a color transition due to a premature interaction between the hydroperoxide and the indicator dye. Accordingly, such a false positive assay decreased the utility and the sensitivity of the test strip to the peroxidatively active substance in the test sample. The indicator reagent composition of the present invention essentially eliminates a premature interaction between the indicator dye and the hydroperoxide. Consequently, the improved stability of the indicator reagent composition increases the sensitivity of the assay such that an accurate and trustworthy assay for a peroxidatively active substance is achieved.
The prior art contains numerous references on the wet phase chemistry and the dry phase chemistry utilized in assaying urine for a peroxidatively active substance. For example, investigators developed wet chemistry assay procedures and dry phase test strip devices for peroxidatively active substances that rely on the enzyme-like catalysis of the peroxidative oxidation of indicator dyes. An example of a wet chemistry assay for a peroxidatively active substance is presented in R. M. Henry, et al., Clinical Chemistry Principles and Techniques, 2nd ed., Harper and Row, pp. 1124-1125 (1974). This wet phase assay procedure employs glacial acetic acid as a buffer, diphenylamine as an indicator dye and hydrogen peroxide. Although such wet phase assays are analytically useful, they nevertheless possess severe disadvantages, including poor reagent stability and inadequate analyte sensitivity. For instance, the reagent solutions used in the wet phase assays rapidly decline in stability, and consequently in sensitivity. Therefore, fresh reagent solutions must be prepared after a few days of storage. The continuous preparation of fresh reagent solutions is time-consuming and uneconomical because costly reagents are wasted.
The preferred method of assaying for a peroxidatively active substance utilizes a dry phase test strip device. A typical dry phase test strip is commercially available from the Diagnostics Division of Miles, Inc. under the trademark HEMASTIX.RTM.. The test strip comprises a test pad, including a porous carrier matrix, such as a paper matrix, impregnated with a buffered mixture of an organic hydroperoxide and an indicator dye, affixed to a plastic strip or handle. The test pad is immersed in a test sample containing hemoglobin, myoglobin, erythrocytes or another peroxidatively active substance, and the test pad develops a blue color. The intensity of the blue color is proportional to the concentration of the peroxidatively active substance in the test sample. By comparing the color developed in the test pad to a standardized color chart, the analyst can determine, quantitatively, the amount of a peroxidatively active substance present in the test sample.
In general, dry phase test strips are more advantageous than the wet phase assays because the test strip format is easier to use, requiring neither the continual preparation of reagents nor the attendant apparatus. In addition, reagent stability is greater in the dry phase test strip, thereby resulting in improved assay accuracy, sensitivity and economy. Notwithstanding that present day test strips for determining peroxidatively active substances are substantially more stable and more sensitive than wet phase assays, present day strips need improvement in the areas of stability and sensitivity. Therefore, it would be a significant advance in the art of diagnostic assays if test strips were even more stable during storage and even more sensitive to peroxidatively active substances. It was towards achieving these improvements that the investigations resulting in the composition, device and method of the present invention were directed.
Several attempts at achieving the above-mentioned goals of increased stability and sensitivity are found in the prior art. For example, in Chemical Abstracts, Vol. 85, p. 186 (1976), a two-dip method of preparing a dry phase test strip containing o-tolidine and phenylisopropyl hydroperoxide is described. In this method, filter paper strips impregnated with ethyl cellulose were dipped into an ethanolic solution comprising an indicator, o-tolidine hydrochloride; polyvinyl pyrrolidone; a surfactant; and sufficient citrate buffer to provide a pH of 3.7. The impregnated filter paper then was dried, and subsequently was dipped into an ethanol-toluene solution containing 1,4-diazobicyclo[2.2.2]octane, phenylisopropyl hydroperoxide and polyvinylpyrrolidone. The investigators desired to stabilize the hydroperoxide with the diazobicyclooctane compound and the polyvinylpyrrolidone, and therefore eliminate a premature interaction with the indicator dye.
Lam, in U.S. Pat. No. 4,071,318, disclosed a composition comprising a hydroperoxide, an indicator dye, and a bicyclic amine borate that is useful in the assay for a peroxidatively active substance. Lam theorized that the bicyclic amine borate complexed with the hydroperoxide, thereby inhibiting the hydroperoxide from interacting with the indictor dye during storage. Therefore, the improved stability of the composition provided more accurate assays for a peroxidatively active substance by reducing the premature oxidation of the indicator dye. However, the amine borates utilized by Lam, such as triethanolamine borate and tri(n-propanol)amine borate were found to hydrolyze upon exposure to environmental humidity and cause a rise in the pH of the indicator reagent composition incorporated in the test pad. The resulting increase in pH caused a decrease in the sensitivity of the assay for a peroxidatively active substance.
H. Steinberg and D. L. Hunter, in the publications, "Preparation and Rate of Hydrolysis of Boric Acid Esters", Ind. and Eng. Chem., 49,2, pp. 174-181 (1974) and "The Hydrolysis of Triisopropanolamine Borate", J. Am. Chem. Soc., 82, pp. 853-859 (1960), disclosed that bicyclic amine borates having pendant methyl groups, like triisopropanolamine borate, hydrolyze substantially more slowly than bicyclic amine borates that do not include pendant methyl groups, like triethanolamine borate. Steinberg et al theorized that the increased stability is attributable to the steric effects introduced by the pendant methyl groups in triisopropanolamine borate. However, the publications of Steinberg et al neither teach nor suggest that sterically-hindered bicyclic amine borates are useful in assays for peroxidatively active substances.
Adams et al, in U.S. Pat. No. 3,252,762, disclosed physically-encapsulating an organic hydroperoxide within a colloidal material, such as gelatin, to stabilize the test strip. Accordingly, when an aqueous test sample contacts the test strip, the gelatin capsules dissolve, thereby freeing the hydroperoxide for an interaction with an indicator dye that is mediated by a peroxidatively active substance. However, the encapsulation process of Adams is time-consuming and requires relatively expensive apparatus and excessive manipulative steps. Each of these prior art disclosures was directed at stabilizing the reagents incorporated into the test pad of the test strip such that the potentially incompatible reagent ingredients, i.e., the hydroperoxide and the indicator dye, would not prematurely interact, and thereby provide a false positive assay or render the test strip less sensitive to a peroxidatively active substance.
Another test strip and method are disclosed in U.S. Pat. No. 3,853,471 to Rittersdorf et al. Rittersdorf described the use of phosphoric acid amides or phosphonic acid amides to stabilize test strips used to assay for peroxidatively active substances. The phosphoric or phosphonic acid amides disclosed by Rittersdorf sufficiently stabilized the hydroperoxide and indicator dye such that the test strips did not become discolored due to a premature interaction between the hydroperoxide and the indicator dye.
Ku, in U.S. Pat. No. 3,411,887, described the elimination of ascorbate interference with reagent compositions that rely on enzymatic oxidizing substances, such as glucose oxidase, by using an ascorbate "trapping system". The "trapping system" utilizes a heavy metal ion that has an oxidation-reduction reduction potential falling between a redox indicator dye and ascorbate. Suitable heavy metal compounds cited as examples include cobalt, iron, mercury and nickel. In addition to the disclosure of Ku, the prior art also discloses that metal ions, such as Co(III), are actually pseudoperoxidases. For example, The Merck Index, 9th ed., p. 311 (1976) discloses the Co(III) acetate is used commercially to catalytically decompose cumene hydroperoxide. In addition, a series of Co(III) complexes to catalytically decompose peroxides are reported by K. Lohs, Monatsber. Deut. Adad. Wiss. Berlin, 8, pp. 657-659 (1966).
U.S. Pat. No. 4,587,220, to Mayambala-Mwanika et al., disclosed the use of a chelated ferric ion to eliminate ascorbic acid and ascorbate ion interference in an assay for a peroxidatively active substance. Mayambala-Mwanika disclosed that a ferric chelate, like the ferric chelate of N-(2-hydroxyethyl)ethylenediaminetriacetic acid (Fe--HEDTA), eliminated ascorbate interference and did not produce a false positive test for the peroxidatively active compound. In accordance with the method of Mayambala-Mwanika, a two-step method of preparing the test device provided an ascorbate-resistant test pad that demonstrated sufficient stability to resist a false positive assay result during storage.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the composition of the present invention has increased stability and therefore imparts increased sensitivity to a test strip used in the detection and measurement of a peroxidatively active substance in a test sample. The method of the present invention utilizes an indicator reagent composition that stabilizes the indicator dye, and therefore essentially eliminates indicator dye interaction with the hydroperoxide until the indicator dye contacts a test sample containing a peroxidatively active substance.
Surprisingly, the method and composition of the present invention essentially eliminate color formation, or other detectable responses, attributable to a premature indicator dye oxidation by the hydroperoxide. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase test strip assay of urine and other test samples for a peroxidatively active substance by utilizing a stable indicator reagent composition that includes a hydrolysis-resistant amine borate having pendate methyl groups and/or ethyl groups.